IRREVERSIBLE INHIBITION OF THE HIV-1 PROTEASE - TARGETING ALKYLATING-AGENTS TO THE CATALYTIC ASPARTATE GROUPS

Irreversible inhibition of the HIV-1 protease by agents that specifica lly alkylate its catalytic aspartate residues is a potentially useful approach for circumventing the evolution of HIV strains that are resis tant to protease inhibitors. Five haloperidol- and two FMOC-based epox ides of differing reactivities have been synthesized and tested as irr eversible inhibitors of the HIV-1 protease (HIV-1 PR). Of these, two t risubstituted epoxides, a cis-1,2- disubstituted epoxide, a 1,1-disubs tituted epoxide, and a monosubstituted epoxide function as irreversibl e inhibitors, but two trans-1,2-disubstituted epoxides do not. The mos t effective of the epoxides (6) inactivates HIV-1 PR with K-inact = 65 mu M and V-inact = 0.009 min(-1). 1,2-Epoxy-3-(p-nitrophenoxy)propane (EPNP), a nonspecific inactivating agent for aspartyl proteases, has been used to validate a protocol for establishing the stoichiometry an d site of protein alkylation. Mass spectrometric analysis of the inact ivated enzyme shows that one molecule of either EPNP or the cyclic 1,2 -disubstituted epoxide 6 is covalently bound per HIV-1 PR dimer. Mass spectrometric sequencing of labeled proteolytic peptides shows that bo th inhibitors are covalently bound to a catalytic aspartate residue. T he covalent binding of three alpha,beta-unsaturated ketone derivatives of haloperidol has been similarly examined. Analysis of HIV-1 PR inac tivated by these agents establishes that they bind covalently to the t wo cysteines and the N-terminal amino group but not detectably to the catalytic aspartate residues. The results indicate that aspartate-targ eted inactivation of HIV-1 PR depends on (a) matching the reactivity o f the alkylating functionality to that of the aspartates, preferably b y exploiting the two-aspartate catalytic motif of the protease to acti vate the alkylating agent, and (b) appropriate positioning of the alky lating functionality within the active site. These requirements are re adily met by a monosubstituted, 1,1-disubstituted, or cyclic cis-1,2-d isubstituted epoxide but not by trans-1,2-disubstituted epoxides or al pha,beta-unsaturated ketones.